ABSTRACT The pathogenic species of Cryptococcus are a major cause of mortality owing to severe infections in immunocompromised as well as immunocompetent individuals. Although antifungal treatment is usually effective, many patients relapse after treatment, and in such cases, comparative analyses of the genomes of incident and relapse isolates may reveal evidence of determinative, microevolutionary changes within the host. Here, we analyzed serial isolates cultured from cerebrospinal fluid specimens of 18 South African patients with recurrent cryptococcal meningitis. The time between collection of the incident isolates and collection of the relapse isolates ranged from 124 days to 290 days, and the analyses revealed that, during this period within the patients, the isolates underwent several genetic and phenotypic changes. Considering the vast genetic diversity of cryptococcal isolates in sub-Saharan Africa, it was not surprising to find that the relapse isolates had acquired different genetic and correlative phenotypic changes. They exhibited various mechanisms for enhancing virulence, such as growth at 39°C, adaptation to stress, and capsule production; a remarkable amplification of ERG11 at the native and unlinked locus may provide stable resistance to fluconazole. Our data provide a deeper understanding of the microevolution of Cryptococcus species under pressure from antifungal chemotherapy and host immune responses. This investigation clearly suggests a promising strategy to identify novel targets for improved diagnosis, therapy, and prognosis. IMPORTANCE Opportunistic infections caused by species of the pathogenic yeast Cryptococcus lead to chronic meningoencephalitis and continue to ravage thousands of patients with HIV/AIDS. Despite receiving antifungal treatment, over 10% of patients develop recurrent disease. In this study, we collected isolates of Cryptococcus from cerebrospinal fluid specimens of 18 patients at the time of their diagnosis and when they relapsed several months later. We then sequenced and compared the genomic DNAs of each pair of initial and relapse isolates. We also tested the isolates for several key properties related to cryptococcal virulence as well as for their susceptibility to the antifungal drug fluconazole. These analyses revealed that the relapsing isolates manifested multiple genetic and chromosomal changes that affected a variety of genes implicated in the pathogenicity of Cryptococcus or resistance to fluconazole. This application of comparative genomics to serial clinical isolates provides a blueprint for identifying the mechanisms whereby pathogenic microbes adapt within patients to prolong disease.

ABSTRACT Patients with cryptococcal meningitis in sub-Saharan Africa frequently relapse following treatment. The natural history and etiology of these recurrent episodes warrant investigation. Here, we used multilocus sequence typing (MLST) to compare the molecular genotypes of strains of Cryptococcus neoformans and Cryptococcus gattii isolated from serial episodes of cryptococcal meningitis that were separated by at least 110 days. The most common MLST genotypes among the isolates were the dominant global clinical genotypes (M5 and M4) of molecular type VNI, as well as the VNI genotypes apparently restricted to southern Africa. In addition, there was considerable genetic diversity among these South African isolates, as 15% of the patients had unique genotypes. Eleven percent of the patients were reinfected with a genetically different strain following their initial diagnosis and treatment. However, the majority of serial episodes (89%) were caused by strains with the same genotype as the original strain. These results indicate that serial episodes of cryptococcosis in South Africa are frequently associated with persistence or relapse of the original infection. Using a reference broth microdilution method, we found that the serial isolates of 11% of the patients infected with strains of C. neoformans var. grubii with identical genotypes exhibited ≥4-fold increases in the MICs to fluconazole. Therefore, these recurrent episodes may have been precipitated by inadequate induction or consolidation of antifungal treatment and occasionally may have been due to increased resistance to fluconazole, which may have developed during the chronic infection.

Fourteen new species in the Latin American Clade (LAC) of the Ceratocystis fimbriata complex recently were distinguished from C. fimbriata sensu stricto largely based on variation in ITS rDNA sequences. Among the 116 isolates representing the LAC, there were 41 ITS haplotypes. Maximum parsimony (MP) analysis of ITS sequences produced poorly resolved trees. In contrast, analyses of matingtype genes (MAT1-1-2 and MAT1-2-1) resolved a single MP tree with branches of high bootstrap and posterior probability support. Four isolates showed intragenomic variation in ITS sequences. Cloning and sequencing of PCR products from a single haploid strain identified two or more ITS sequences differing at up to 16 base positions and representing two described species. Isolates from introduced populations that appeared to be clonal based on microsatellite markers varied at up to 14 bp in ITS sequence. Strains of seven Brazilian ITS haplotypes and an isolate from Ipomoea batatas (on which the species name C. fimbriata was based) were fully interfertile in sexual crosses. These analyses support three phylogenetic species that differ in pathogenicity: C. platani, C. cacaofunesta and C. colombiana. Five ITS species (C. manginecans, C. mangicola, C. mangivora, C. acaciivora, C. eucalypticola) appear to be ITS haplotypes that have been moved from or within Brazil on nursery stock. The taxonomic status of other species delineated primarily by ITS sequences (C. diversiconidia, C. papillata, C. neglecta, C. ecuadoriana, C. fimbriatomima, C. curvata) needs further study, but they are considered doubtful species.

Commercial plantations of Eucalyptus species have been established in South China, especially during the past 20 years, to meet the needs of a rapidly growing national economy. As part of a survey of fungal diseases affecting Eucalyptus species in South China, Ceratocystis species were collected from Eucalyptus plantations in the GuangDong Province. The aims of this study were to identify these Ceratocystis isolates and to test their pathogenicity to Eucalyptus. The most aggressive isolates were also used to screen different species and clones of Eucalyptus for susceptibility to infection under field conditions. The fungi were identified based on morphology and through comparisons of DNA sequence data of the ITS, partial β-tubulin and TEF-1α gene regions. Morphological and DNA sequence comparisons showed that isolates collected from Chinese Eucalyptus plantations represent two species, C. acaciivora in the C. fimbriata s.l. species complex and a previously undescribed species belonging to the C. moniliformis s.l. species complex, for which the name C. chinaeucensis sp. nov. is provided. In pathogenicity trials, both C. acaciivora and C. chinaeucensis gave rise to lesions on wounded Eucalyptus trees, and the former fungus was most pathogenic. Differences were also observed in the responses of different Eucalyptus clones to inoculation and this could be useful in reducing disease, if C. acaciivora should emerge as a pathogen in the future.

BACKGROUND:The genus emmonsia contains three species that are associated with human disease. Emmonsia crescens and Emmonsia parva are the agents that cause adiaspiromycosis, and one human case of Emmonsia pasteuriana infection has been described. We report a fungal pathogen within the genus emmonsia that is most closely related to E. pasteuriana in human immunodeficiency virus (HIV)-infected adults in South Africa.METHODS:Between July 2008 and July 2011, we conducted enhanced surveillance to identify the cause of systemic, dimorphic fungal infections in patients presenting to Groote Schuur Hospital and other hospitals affiliated with the University of Cape Town, Cape Town, South Africa. DNA sequencing was used to identify pathogenic fungi.RESULTS:A total of 24 cases of dimorphic fungal infection were diagnosed, 13 of which were caused by an emmonsia species. All 13 patients were HIV-infected, with a median CD4+ T-cell count of 16 cells per cubic millimeter (interquartile range, 10 to 44), and all had evidence of disseminated fungal disease. Three patients died soon after presentation, but the others had a good response to a variety of antifungal agents and antiretroviral therapy. Phylogenetic analysis of five genes (LSU, ITS1-2, and the genes encoding actin, β-tubulin, and intein PRP8) revealed that this fungus belongs in the genus emmonsia and is most closely related to E. pasteuriana.CONCLUSIONS:The findings suggest that these isolates of an emmonsia species represent a new species of dimorphic fungus that is pathogenic to humans. The species appears to be an important cause of infections in Cape Town.

Eucalyptus trees, mostly native to Australia, are widely planted in the tropics and Southern Hemisphere for the production of wood and pulp. Worldwide surveys of diseases on these trees have yielded a large collection of Ceratocystis isolates from dying trees or from wounds on their stems. The aim of this study was to characterise these isolates and to consider their relatedness to each other. Culture appearance, morphological features and a distinctive fruity odour in all cultures were typical of species in the Ceratocystis fimbriatasensu lato (s. lat.) complex. Phylogenetic analyses of sequences for the combined ITS, βt-1 and TEF1-α gene regions revealed a genetically diverse group of isolates residing in a single large clade, that were distinct from all other species in the C. fimbriatas. lat. complex. Based on morphology and phylogenetic inference, the Eucalyptus isolates are recognised as closely related. The South African isolates are described here as a new species, C. eucalypticola.

Mangifera indica, a disease known as mango blight, murcha or seca da mangueira in Brazil, is caused by the canker wilt pathogen Ceratocystis fimbriata sensu lato. It is also closely associated with infestation by the non-native wood-boring beetle Hypocryphalus mangiferae (Coleoptera: Scolytinae). The aim of this study was to characterize Ceratocystis isolates obtained from diseased mango trees in Brazil. Identification was based on sequence data from ITS1+5.8S+ITS2 rDNA, part of the Beta-tubulin 1 gene, and part of the Transcription Elongation Factor 1-alpha gene. The Brazilian isolates grouped in two well defined and unique clades within C. fimbriata s.l. These were also distinct from C. manginecans, which causes a similar disease associated with H. mangiferae in Oman and Pakistan. Based on sequence comparisons and morphological characteristics, isolates representing the two phylogenetic clades are described as C. mangicola sp. nov. and C. mangivora sp. nov.

Pruning (singling) is a common silvicultural practice in commercial Acacia plantations because these trees tend to have multiple stems. The wounds resulting from pruning are susceptible to infection by pathogens. Ceratocystis acaciivora and Lasiodiplodia theobromae have been shown recently to be important pathogens of A. mangium in Indonesia, where they are commonly associated with wounds on trees. The aim of this study was to determine the impact of different wound types on infection of A. mangium and A. crassicarpa by these two pathogens. Isolates of C. acaciivora and L. theobromae, found to be the most pathogenic in a prior study, were used to inoculate pruning wounds. Results showed that pruning conducted in a manner to reduce stem damage, resulted in lower levels of fungal infection. Where pruning resulted in tearing of the bark, there were higher levels of infection and disease occurred even without artificial inoculation. Inoculation of pruning wounds with C. acaciivora and L. theobromae showed that both fungi have the potential to cause disease. However, C. acaciivora was most virulent. Results of this study showed conclusively that careful pruning will result in lower levels of disease in young A. mangium and A. crassicarpa plantations.

ABSTRACT Cryptococcus neoformans is the most common cause of meningitis among adult South Africans with HIV infection/AIDS. Widespread use of fluconazole for treatment of cryptococcal meningitis and other HIV-associated opportunistic fungal infections in South Africa may lead to the emergence of isolates with reduced fluconazole susceptibility. MIC testing using a reference broth microdilution method was used to determine if isolates with reduced susceptibility to fluconazole or amphotericin B had emerged among cases of incident disease. Incident isolates were tested from two surveillance periods (2002-2003 and 2007-2008) when population-based surveillance was conducted in Gauteng Province, South Africa. These isolates were also tested for susceptibility to flucytosine, itraconazole, voriconazole, and posaconazole. Serially collected isolate pairs from cases at several large South African hospitals were also tested for susceptibility to fluconazole. Of the 487 incident isolates tested, only 3 (0.6%) demonstrated a fluconazole MIC of ≥16 μg/ml; all of these isolates were from 2002-2003. All incident isolates were inhibited by very low concentrations of amphotericin B and exhibited very low MICs to voriconazole and posaconazole. Of 67 cases with serially collected isolate pairs, only 1 case was detected where the isolate collected more than 30 days later had a fluconazole MIC value significantly higher than the MIC of the corresponding incident isolate. Although routine antifungal susceptibility testing of incident isolates is not currently recommended in clinical settings, it is still clearly important for public health to periodically monitor for the emergence of resistance.

Ceratocystis fimbriata sensu lato includes a large number of plant and especially tree pathogens. In Colombia, isolates of this fungus cause a serious canker-stain disease on coffee as well as other fruit trees. Large collections of these isolates have been shown to occur in two distinct phylogenetic lineages based on ITS sequence comparisons. The aim of this study was to compare representatives of these two groups of isolates from coffee, citrus, cacao and native trees in Colombia, based on morphology and DNA-sequences for three gene regions. Host-specificity of the fungus was also considered. Representatives of the two groups of isolates were morphologically distinct and could be distinguished based on DNA sequence comparisons. They are also distinct from other species in the C. fimbriata sensu lato species complex and the sweet potato pathogen C. fimbriata sensu stricto and are provided with the names C. colombiana sp. nov and C. papillata sp. nov. There was no evidence for host-specificity amongst isolates of these two fungi that collectively represent a serious threat to coffee production in Colombia.

Ceratocystis fimbriata sensu lato (s.l.) is an important pathogen of Eucalyptus. Pathogenicity of isolates has typically been evaluated by inoculating seedlings under greenhouse conditions. It is, however, not clear how accurately this reflects pathogenicity under field conditions. In this study, five techniques to potentially screen C. fimbriata isolates for their relative pathogenicity to Eucalyptus were compared. These included: in vitro growth comparisons on artificial media; inoculations on apples; inoculation on Eucalyptus seedlings in a greenhouse; inoculations on Eucalyptus bolts freshly cut from stems of young trees; and field inoculations on young trees. Eight isolates of C. fimbriata s.l. collected from various areas in South Africa were used. There was considerable variation in growth in culture and aggressiveness of the eight isolates. Field inoculations on young trees were best correlated with inoculations of bolts (r = 0.76). Lower correlation coefficients were obtained with seedlings (r = 0.59), apple inoculations (r = 0.56), and in vitro colony growth (r = 0.42). Inoculation of bolts provides a rapid and reliable method to screen isolates of C. fimbriata s.l. for pathogenicity to Eucalyptus.

Species of Ceratocystis commonly infect wounds on trees. In this study, artificially induced wounds were made on the stems of Eucalyptus , Schizolobium and Terminalia trees in Ecuadorian lowland forests, in an effort to determine the presence of Ceratocystis spp. in that environment. Species belonging to the C. fimbriata sensu lato ( s.l. ) complex and others in the C. moniliformis s.l. complex were collected. Phylogenetic analyses for both major groups in Ceratocystis using three gene regions (ITS, β -tubulin and EF1- α ) revealed three distinct clades in the C. fimbriata s.l. complex and two in the C. moniliformis s.l. complex. Isolates in the three clades representing the C. fimbriata s.l. complex represent morphologically distinct species that are described here as C. curvata sp. nov. , C. ecuadoriana sp. nov. and C. diversiconidia sp. nov . Isolates in one of the two clades in the C. moniliformis species complex represented C. moniliformis sensu stricto ( s.s. ) and the other was of a species with a distinct morphology that is described here as C. sublaevis sp. nov.

The genus Ceratocystis includes many important tree pathogens and agents of sap stain. These fungi have a global distribution and commonly infect wounds on trees. During a survey of wound-infecting pathogens in the genus Ceratocystis on plantation-grown Acacia mangium trees in Indonesia, several isolates resembling Ceratocystis moniliformis sensu lato (s.l.) were obtained. The aim of this study was to identify these isolates and to test their pathogenicity on commercially grown Acacia spp. in the country. Use was made of morphology and comparisons of DNA sequence data for the ITS, β-tubulin, and transcription elongation factor 1-α gene regions to identify the isolates. Three previously undescribed species in the C. moniliformis s.l. species complex were identified and are described here as C. inquinans sp. nov., C. sumatrana sp. nov., and C. microbasis sp. nov. Pathogenicity trials on Acacia mangium and A. crassicarpa in the greenhouse and in the field indicated that all three species have the potential to infect A. mangium and A. crassicarpa, although they are not considered important pathogens.

Species of Ceratocystis are well-known wound related pathogens of many tree species, including commercially planted Acacia spp. Recently, several Ceratocystis isolates were collected from wilting A. mangium in plantations in Indonesia. The aim of this study was to identify these Ceratocystis isolates and to investigate their ability to cause disease on two plantation-grown Acacia spp. using greenhouse and field inoculation experiments. For identification, morphological characteristics and comparisons of DNA sequence data for the ITS, β-tubulin and TEF 1-α gene regions, was used. Ceratocystis isolates were identified as C. manginecans, a serious pathogen of mango trees in Oman and Pakistan and a previously undescribed species, described here as C. acaciivora sp. nov. Both fungi produced significant lesions in inoculation experiments on A. mangium and A. crassicarpa, however, C. acaciivora was most pathogenic suggesting that this fungus is the primary cause of the death of trees under natural conditions.

Styrax benzoin trees, native to the island Sumatra, Indonesia are wounded to produce resin that is collected and burned as incense. These wounds on trees commonly develop into expanding cankers that lead to tree death. The aim of this study was to consider whether Ophiostomatoid fungi, typically associated with wounds on trees might be associated with resin harvesting on S. benzoin. Samples were collected from the edges of artificially induced wounds, and particularly where cankers and staining of the vascular tissue was evident. Tissue samples were incubated in moist chambers and carrot baiting was also used to detect the presence of Ceratocystis spp. Fruiting structures with morphology typical of species in the C. fimbriata s.l. species complex and species in the anamorph genus Thielaviopsis were found, on both the incubated wood and the carrot baits. DNA sequences were generated for the Internal Transcribed Spacer regions 1 and 2 including the 5.8S rRNA gene, part of the beta-tubulin and the Transcription Elongation Factor 1-alpha gene regions. These data were compared with those of other species in the C. fimbriata s.l. species complex and Thielaviopsis using phylogenetic analysis. Morphology of the isolates in culture as well as phylogenetic inference showed that the Thielaviopsis sp. present on the wounds was T basicola. The Ceratocystis sp. from S. benzoin represents a new taxon in the C. fimbriata s.l. complex described here as C. larium sp. nov.

Species of Ceratocystis Ellis and Halstead s.l. include important plant pathogens such as C. albifundus Morris, De Beer, and M. J. Wingfield that causes a serious wilt disease of non-native, plantation-grown Acacia mearnsii De Wild. trees in Africa. The aim of this study was to identify the insects associated with C. albifundus in South Africa and to consider the means by which the pathogen spreads. Insects were collected weekly for 77 wk in a native ecosystem using modified pitfall traps. Trapped insects were identified, and fungi were isolated using carrot baiting and by plating them onto malt extract agar. Fungi were identified using morphological characteristics and DNA sequence comparisons. Three different nitidulid (Coleoptera: Nitidulidae) beetles, Brachypeplus depressus Erichson, Carpophilus bisignatus Boheman, and Ca. hemipterus L, were collected, of which the most common were the Carpophilus spp. Two Ceratocystis spp., namely G albifundus and C. oblonga R.N. Heath and Jolanda Roux, were isolated from all three insect species. Insect numbers and fungal isolates decreased significantly in the colder months of the year. Of the two Ceratocystis spp., C. oblonga was most abundant, occurring on 0.5% of the Carpophilus spp. C. albifundus was isolated from 1.1% of the Brachypeplus individuals and from 0.01% of the Carpophilus individuals. This study presents the first record of insects associated with C. albifundus and C. oblonga and provides an indication of environmental influences on fungal and insect populations, which could contribute to future disease management.